P-013: VOXELOTOR REDUCES SICKLE RED BLOOD CELL-MEDIATED HYPOXIC MICROVASCULAR OCCLUSION AS MEASURED BY OCCLUSIONCHIP IN VITRO MICROFLUIDIC ASSAY
Authors: SEKYONDA Z., OSHABAHEEBWA S., MONCHAMP K., SHEEHAN V., GURKAN U.
Journal: HemaSphere (2022)

Purpose: Sickle cell disease (SCD) is an inherited blood disorder caused by a single gene mutation that results in the production of abnormal hemoglobin, sickle hemoglobin (HbS). Under deoxygenation, HbS tends to polymerize, resulting in deformed, rigid, and fragile red blood cells (RBCs). These RBC alterations result in vaso-occlusion and hemolysis. The US FDA has recently approved voxelotor, an oxygen affinity modulating drug for the treatment of SCD. When voxelotor binds to HbS, it stabilizes HbS in an oxygenated configuration, which reduces HbS polymerization and RBC sickling [1]. However, the overall impact of voxelotor on RBC capacity for vaso-occlusion has not been examined. Here we use a microfluidic device, the OcclusionChip [2], to investigate the effect of voxelotor on the RBC capacity for occlusion in SCD. Materials and methods: Venous blood samples were collected in EDTA from subjects with homozygous HbSS and healthy volunteers (HbAA) under an IRB-approved protocol. OcclusionChip devices were fabricated using soft lithography protocols [2]. RBCs were suspended in PBS at 20% hematocrit. To create Vox + samples, HbSS containing RBCs at 20% hematocrit were mixed with 67mg/ml voxelotor in 100% dimethyl sulfoxide (DMSO) to a final concentration of 600 µmol/L voxelotor and incubated at 37 C0 for 1 hour. RBCs were perfused through the OcclusionChip with a constant inlet pressure, followed by a PBS wash and microscopy. For hypoxia experiments, blood was deoxygenated in OcclusionChip (pO2 ~45 mmHg) [2]. The Occlusion Index (OI), representing the overall microcapillary network percentage occlusion, was quantified as previously described [2]. Data are reported as mean ± standard deviation (SD). Results: Using the OcclusionChip, we analyzed RBC-mediated microvascular occlusion for healthy (HbAA) and HbSS samples under normoxia and hypoxia in comparison to voxelotor treated HbSS samples under hypoxia (Figure 1A). There was no statistical difference between the OI values for HbAA samples under normoxic versus hypoxic conditions (P = 0.50). The OI values for HbSS samples were significantly elevated under hypoxia in comparison to normoxia (P = 0.007) (Figure 1B). However, when HbSS samples were treated with Voxelotor, the OI values significantly decreased under hypoxia by 52.6% ± 10.47% (mean ± SD, P = 0.010) (Figure 1B) but were still significantly greater than the OI of HbAA (P = 0.001) (Figure 1B). Conclusion: Our findings suggest that hypoxia alters the deformability of SCD RBCs but not healthy RBCs. This is likely due to HbS polymerization during deoxygenation. Under hypoxic conditions, HbSS RBCs treated with voxelotor exhibited significantly less occlusion in the Occlusion Chip, suggesting that voxelotor may reduce vasoocclusion in vivo. Voxelotor reduced OI to different degrees in RBCs from different individuals, indicating inter-patient variability in response to voxelotor for this aspect of SCD pathophysiology. Correlation between this in vitro test and in vivo response to voxelotor is warranted, as well as investigations with other novel SCD therapies, to validate the OI’s utility as a functional indicator for evaluating the efficacy of novel therapies in preventing microcapillary occlusions. 1.Elliott et al., NEJM, 2019,381:509-519 2.Man et al., LabChip, 2020, 20, 2086-2099.Figure 1. OcclusionChip: standardized microfluidic assessment of RBC-mediated microcapillary occlusion in Healthy (HbAA), SCD (HbSS), and SCD treated with voxelotor. (A) Occlusion Chip device for interrogation of RBC-mediated microcapillary occlusion. (B) The authors do not declare any conflict of interest